Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense sequences to enhance one or more properties such as for example nuclease resistance. One such group of chemical modifications includes bicyclic nucleosides wherein the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring thereby forming a bicyclic ring system. Such bicyclic nucleosides can have various names including BNAs and LNAs for bicyclic nucleic acids or locked nucleic acids respectively.
Various BNAs have been prepared and reported in the patent literature as well as in the scientific literature, see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Wengel et al., PCT International Application WO 98-DK393 19980914; Singh et al., J. Org. Chem., 1998, 63, 10035-10039, the text of each is incorporated by reference herein, in their entirety. Examples of issued US patents and published applications that describe various BNAs include for example: U.S. Pat. Nos. 7,053,207, 6,770,748, 6,268,490 and 6,794,499 and published U.S. applications 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114 and 20030082807; the text of each is incorporated by reference herein, in their entirety.
BNAs have also been reported in the scientific literature having the L configuration (α-L-BNA or α-L-LNA) wherein most of these α-L-BNAs have been studied in oligomeric compounds, see for example: Gaubert, G. et al., Nucleosides Nucleotides Nucleic Acids 2003, 22, 1155-1157; Kumar et al., J. Org. Chem. 2006, 71, 4188-4201; Fluiter et al., ChemBioChem, 2005, 6, 1-6; Arzumanov et al., Oligonucleotides, 2003, 13, 435-453; Frieden et al. Nucleic Acids Res., 2003, 31(21), 6365-6372; Sorensen et al., J. Am. Chem. Soc. 2002, 124, 2164-2176; Petersen et al., J. Am. Chem. Soc. 2001, 123, 7431-7432; Rajwanshi et al., Angew Chem Int Ed Engl 2000, 39, 1656-1659; Rajwanshi et al., J. Chem. Commun. 1999, 1395-1396; and U.S. Pat. No. 7,053,207; the text of each is incorporated by reference herein, in their entirety.
In a recent in vivo study with LNAs in mice, hepatotoxicity was reported. See, e.g., Swayze et al., Antisense oligonucleotides containing locked nucleic acid improve potency but cause significant hepatotoxicity in animals, Nucl. Acids Res., 2007, 35(2), 687-700.
One carbocyclic BNA has been previously reported (Frier et al., Nucleic Acids Research, 1997, 25 (22), 4429-4443) having a 4′-(CH2)3-2′ bridge. This modification was reported to drop the Tm about 2.5° C. per modification for a poly T oligomer having a BNA T nucleoside in place of one or more T nucleosides when hybridized to complementary RNA. The BNA having the 4′-(CH2)3-2′ bridge as reported by Frier et al., and an the alkenyl bridged analog (4′-CH═CH—CH2-2′) has also been reported to have increased stability with an increased Tm of from about 2.5 to 5.0° C. per modification (see Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). Carbocyclic bicyclic nucleosides and oligonucleotides prepared therefrom have also been recently reported by Srivastava et al., J. Am. Chem. Soc. 2007, 129, 8362-8379.
Consequently, there remains a long-felt need for agents that specifically regulate gene expression via antisense mechanisms. Disclosed herein are α-L-carbocyclic BNA's that are useful for enhancing one or more properties of the oligomeric compounds they are incorporated into such as nuclease resistance. The oligomeric compounds are expected to be useful for preparing antisense compounds for use in for modulating gene expression pathways, including those relying on mechanisms of action such as RNaseH, RNAi and dsRNA enzymes, as well as other antisense mechanisms based on target degradation or target occupancy. One having skill in the art, once armed with this disclosure will be able, without undue experimentation, to identify, prepare and exploit antisense compounds for these uses.